N-terminal peptides of LAG-3 (WT, V20A, and L14Q/V20A) were quantified by PRM

The abundance of N-terminal fragments starting from V21 (pV21–W33), S22 (pS22–W33), S23 (pS23–W33), and G24 (pG24–W33) along with three control peptides (Ctrl1, pH81–Y95; Ctrl2, pR106–L118; Ctrl3, pH113–L123) were measured by parallel reaction monitoring (PRM), an MS/MS-based targeted quantification method using high-resolution MS. Targeted MS/MS scans were acquired by a time-scheduled inclusion list, and time alignment and relative quantification of the transitions of three biological replicates were performed with PinPoint version 1.4 (Thermo Fisher Scientific).